Lipid peroxidation of rat liver microsomal fractions was monitored by its low-level cheniiluniinescence in preparations from controls and vitamin-E-deficient animals. Measurements were made (a) of the duration of the lag phase T~ after initiation with NADPH/iron-ADP and (b) of the slope of the chemiluminescence increase.In microsomes with normal vitamin E (a-tocopherol) level the lag phase T,, was substantially increased by ascorbate; in contrast, even an enhanced peroxidation was observed with ascorbate in vitamin-E-deficient microsomes. Therefore, the ascorbate-mediated protection of microsomal membranes against lipid peroxidation is dependent on vitamin E in the membrane. In vitamin E deficiency the pro-oxidant effect of ascorbate was abolished when glutathione (GSH) was present.Likewise, GSH does not prolong the lag phase zo in vitamin E deficiency. However, GSH (but not cysteine) exerts an antioxidant effect both in controls and in vitamin E deficiency by decreasing the slope of the chemiluminescence increase during lipid peroxidation. The involvement of GSH in an enzyme-dependent mechanism is suggested.Vitamin E is regarded as the major lipid-soluble antioxidant, preventing oxidative attack of membrane lipids and other membrane-associated compounds (see [ 11 for review). The initiating process by a primary radical R' leads to lipid radicals L', which then add oxygen in a diffusion-limited reaction. The resulting lipid peroxyl radicals continue a chain process.(1) (2) Chain-breaking antioxidants shorten the length of the chain reaction by trapping the peroxyl radical LOO'. Vitamin E has been shown to be a very effective scavenger of these radicals [2, 31, whereas it does not react at comparable rates with carbon-centered radicals [4].Ascorbate [5 -71 and glutathione (GSH) [8 -151 have been described to be involved in several types of protective mechanism. In ESR studies regeneration of vitamin E from the cr-chromanoxyl radical by GSH and vitamin C was detected [lo, 161. In experiments investigating the effect of vitamin E and vitamin C on methyl linoleate peroxidation, vitamin E was kept in the reduced state as long as there was vitamin C present [17]. In addition to this synergistic effect of vitamin C with vitamin E, a direct chain-breaking activity of vitamin C was also reported [17]. In human blood plasma the oxidation of protein sulfhydryl groups is one of the first events when peroxyl radicals are generated [I 81, thus a chain-breaking
The reaction of superoxide with reduced glutathione (GSH) was studied with two 0;-producing systems : xanthine oxidase using xanthine or acetaldehyde as substrates and, secondly, quinol autoxidation.The capability of GSH to quench superoxide radicals was detected by lowered 0;-mediated cytochrome c3+ reduction. The formation of the oxidation products, glutathione disulfide (GSSG) and glutathione sulfonate (the latter at levels of about 6 -15 % compared to GSSG), was dependent on the 0; production and was inhibited by superoxide dismutase. The presence of GSH together with an 06-producing system led to an extra uptake of oxygen, which was also depressed by superoxide dismutase. The observed O2 uptake was accounted for by the formation of GSSG and GSO; from GSH; the data are in accordance with a mechanism involving thiyl radicals.Low-level chemiluminescence measurement indicated the formation of excited oxygen species. The intensity of photoemission was dependent on the GSH concentration and on the 0 6 production rate. Chemiluminescence was inhibited by superoxide dismutase and also by glutathione peroxidase, but not by catalase or OH. quenchers. Spectral analysis and the effects of 1,4-diazabicyclo[2.2.2]octane and sodium azide indicated the contribution of singlet molecular oxygen to the light emission. It is suggested that singlet oxygen results from an intermediate oxygen addition product such as a glutathione peroxysulphenyl radical.The superoxide anion radical 0; (and its protonated form HO;) is a radical of biological importance known to be generated in a large number of enzymatic and non-enzymatic reactions. Though cellular defense mechanisms against cell toxicity of 0 6 or HOi and further products of electron transfer to oxygen (H202, OH') are effective, these may be overwhelmed in oxidative stress with excessive formation of 0;. As a consequence, pronounced effects on the cellular glutathione (GSH) status have been described [l -31. The loss of GSH is explainable by GSH-peroxidase-mediated reduction of H20z, the latter being the product of HOi dismutation. However, the possibility that GSH also reacts directly with 0; or HO; may be considered. McNeil et al.[4] described the inhibition effect of GSH on superoxide-dependent epinephrine oxidation and on the oxidation of dianisidine and suggested that GSH may play a major role in controlling 0; concentrations in the cell. Moreover, in recent work [3] we described the effect of lowered GSH content on menadione-induced chemiluminescence of perfused rat liver; the light emission was lowered when the GSH level was decreased, thus suggesting that GSH could play a role in the generation of light-emitting species such as singlet molecular oxygen. Similar effects were found with microsomes during redox cycling of the menadione-GSH conjugate [3].Abbreviations. DABCO, 1,4-diazabicyclo[2.2.2]octane; GSH, glutathione, reduced form; GSSG, glutathione disulfide; menadione-GSH conjugate, 2-methyl-3-glutathionyl-1 ,4-naphthoquinone ; HPLC, high-pressure liquid chromatograph...
1. Oxygenation of isolated hepatocytes leads to an increased emission of low level chemiluminescence and to an accumulation of malondialdehyde, both occurring after a lag phase of about 20 -40 min.2. Spectral analysis of oxygen-induced chemiluminescence of isolated hepatocytes showed three bands at 460,560 and 640 nm, with two shoulders at 525 and 61 5 nm. Singlet molecular oxygen, formed during the free radical process accompanying lipid peroxidation, is identified as the main source of light emission, on the basis of comparison with spectra of singlet oxygen produced in chemical systems [Khan, A. U. und Kasha, M. (1963) J . Am. Chem. Soc. 92,3. Hepatocytes from phenobarbital-pretreated rats, or glutathione-depleted hepatocytes showed a threefold increase in both maximal chemiluminescence intensity and malondialdehyde accumulated, as compared with control cells, whereas the lag phase was not modified by the pretreatments.4. Glutathione-depleted hepatocytes did not show any increase in spontaneous lipid peroxidation as reflected by either malondialdehyde accumulation or chemiluminescence. A dissociation between both parameters was observed on addition of dithioerythritol : chemiluminescence intensity decreased while the malondialdehyde content remained unaltered, 5. It is concluded from these experiments that low-level chemiluminescence emitted from hepatocytes at wavelengths beyond 600 nm ('red band') monitors the steady-state concentration of singlet molecular oxygen, providing a useful tool to examine oxygen-dependent radical damage. Continuous monitoring of singlet oxygen levels affords an advantage over parameters measuring accumulative effects.Low-level chemiluminescence is related to the free radical processes that accompany oxidation of lipids, and specifically to the generation of excited species derived from the interaction of free radicals [I -51. Spontaneous chemiluminescence arising from organs, cells and organelles can give account of endogenous oxidation reactions involving lipids within a physiological frame. However, the count rate for spontaneous chemiluminescence is extremely low, making difficult its characterization. Therefore, efforts are usually made to enhance this spontaneous chemiluminescence by peroxidative conditions, such as NADPH-supplemented microsomal fractions in the absence of autoxidizable substrates [6,7], addition of Fez + or organic hydroperoxides to mitochondria1 preparations [8,9], perfusion of organs with organic hydroperoxides [lo, 1 I] or exposure to hyperbaric oxygen conditions [5].In the present study, we describe the characteristics of chemiluminescence of isolated hepatocytes, as observable in the widely used incubation condition with Krebs-Henseleit buffer, and this is extended to certain conditions, such as glutathione depletion and induction of microsomal enzymes, that favour lipid peroxidation. Hepatocytes, isolated with a technique associated with low peroxidative damage, provide a suitable biological source to evaluate the oxidative events through the chemilu...
Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen ('03 generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E.coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet-oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D,O led to an increase in DNA damage. 9,10-Bis(2-ethylene)anthracene disulfate was used as a chemical trap for '0, quantitation by HPLC analysis of the endoperoxide formed.Singlet oxygen; DNA damage; Plasmid pBR322; DNA
Menadione elicits low-level chemiluminescence (h > 620 nm) associated with redox cycling of the quinone in mouse hepatic postmitochondrial fractions. This photoemission is suppressed when the animals are fed a diet containing the anticarcinogenic antioxidant, 2[3]-(tert-butyl)-4-hydroxyanisole (BHA), which leads to a 13-fold increase in NAD(P)H:quinone reductase (EC 1.6.99.2). Inhibition of the enzyme by dicoumarol completely abolishes the protective effect of BHA treatment and leads to higher chemiluminescence, reaching similar photoemission for BHA-treated and control animals. These findings indicate that the two-electron reduction promoted by quinone reductase prevents redox cycling and that BHA protects against reactive oxygen species by elevating the activity of this enzyme. Reactive oxygen species Dietary antioxidant DT-diaphorase Low-level chemiluminescence DicoumarolQuinone redox cycling
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